PROJECT SUMMARY Hepatocellular carcinoma (HCC) is the 2nd most common cause of cancer-related deaths worldwide. Liver transplantation (LT) is the only curative therapy for HCC patients with unresectable, non-metastatic disease who meet strict radiologic tumor size and number criteria (Milan criteria). Eligible candidates undergo a finite observation period (>6 months) that allows for an assessment of tumor biology, but which inherently risks HCC progression and wait-list dropout in 15-20% of patients after 1 year. Despite these selection practices, post-LT HCC recurrence plagues up to 20% of patients, and is a major cause of allograft loss and patient mortality. There is a dire need for non-invasive biomarkers capable of dynamic monitoring of tumor biology to better balance the risk of wait-list dropout and post-LT recurrence, and allowing for improved prioritization of HCC patients to receive scarce liver allografts. This proposal aims to develop an integrated blood-based analysis (i.e., NanoVelcro vimCTC Assay for detecting vimentin+ circulating tumor cells [CTCs] and HCC CTC-RNA Assay for HCC- specific RNA signatures) for wait-listed HCC patients, to identify patients most suitable for LT. The integrated assay will provide a novel approach to study both phenotypic and molecular characteristics of HCC CTCs. Using an HCC-specific multi-marker capture cocktail and optimized immunocytochemistry (ICC) staining protocol, the proposed NanoVelcro vimCTC Assay is capable of identifying a subpopulation of HCC CTCs with vimentin expression (named vimCTC, DAPI+/CK+/CD45-/vimentin+). This subpopulation is associated with increased recurrence in the subset of clinically indistinguishable early-stage patients undergoing curative-intent treatment. The HCC CTC-RNA Assay was developed by combining CTC isolation with Click Chip, featuring click chemistry-mediated cell capture and disulfide-cleavage cell release, with downstream RNA expression profiling of the purified CTCs with NanoString's nCounter platform. This allows for accurate quantification of a panel of HCC CTC-derived mRNA markers in a non-invasive manner. The resulting vimCTC counts and mRNA profiles hold great promise to augment the ability of the current LT candidate selection algorithm. The proposed research will be implemented via Specific Aim 1a: Conducting a retrospective study in banked blood samples using NanoVelcro vimCTC Assay to refine the association between vimCTC counts and post- LT recurrence/wait-list dropout.; Specific Aim 1b: Conducting a prospective study on freshly collected blood samples to determine association between vimCTC counts and post-LT recurrence/wait-list dropout; and Specific Aim 2: Conducting a prospective study on freshly collected blood samples to determine the association between HCC CTC-RNA Assay and post-LT recurrence/wait-list dropout. The central hypothesis evaluated will be that baseline and longitudinal changes in vimCTCs and aggressive RNA signatures will significantly improve the ability of current clinicoradiologic LT selection criteria in predicting post-LT recurrence and wait-list dropout, paving the way for tumor-biology based HCC LT candidate selection practices.